Hypoxia from birth in immature rabbits increases the tolerance of isolated hearts to ischemia compared with age-matched normoxic controls. We determined if this increased tolerance was due to alterations in endogenous nitric oxide production. To test the hypothesis, rabbits (n=8/group) were raised from birth in a normoxic or hypoxic environment for 7-10 days and the hearts perfused with bicarbonate buffer. A nitric oxide donor S-nitrosoglutathione (GSNO) or a nitric oxide synthase inhibitor NG-nitro-L-arginine (L-NAME) was added 15 min prior to a global ischemic period of 30 min, followed by 35 min reperfusion. GSNO (10 micromole/L) increased the recovery of left ventricular developed pressure in normoxic hearts to values not different from hypoxic hearts but had no effect on recovery of developed pressure in hypoxic hearts. L-NAME (200 micromole/L) completely abolished the cardioprotective effect of chronic hypoxia but had no effect on cardioprotection in normoxic hearts. To determine if altered nitric oxide synthase (NOS) gene expression correlated with increased tolerance to ischemia constitutive NOS (NOS3) and inducible NOS (NOS2) transcripts levels were determined by Northern analysis. Hypoxia from birth increased NOS3 expression by 238% of normoxic values when normalized to the internal control, alpha-tubulin. NOS2 was not found in normoxic or hypoxic hearts. Release of nitrite, nitrate, and cGMP from hypoxic hearts as well as tissue cGMP was greater than in normoxic hearts. These data indicate that hypoxia from birth in immature hearts elevates expression of constitutive nitric oxice synthase which increases tolerance to ischemia.